FIGURE 5.

RNA1 mutations affect plasmid copy-number. (A) Schematic linear map of the pVG1 construct. The orange section is from the vector backbone, which includes a ColE1 origin of replication for E. coli (but not for S. aureus), an ampicillin resistance cassette for E. coli and a chloramphenicol resistance cassette for S. aureus. The green section was cloned from pSA564, and includes (from left to right) a truncated rep1_N gene, the rac gene, the RNA1 gene, and the repA_N gene. The location of the rac promoter is unknown. Further details about the pSA564 insert can be found in Supplementary Figure 1. (B) The copy number of pVG1 and its mutant derivatives was estimated by plating on medium containing increasing concentrations of chloramphenicol. Growth at higher concentrations indicate higher chloramphenicol resistance gene dosage (i.e., higher plasmid copy number). The experiment was carried out in the PR02 (a RN4220 derivative) host strain, and plasmid replication is therefore whole dependent on cis-produced RepA. Details of the mutations in pVG1 can be found in Table 2.